Transarterial chemoembolization (TACE) combined with sorafenib (TACE-S) or apatinib (TACE-A) is used in the treatment of hepatocellular carcinoma (HCC). However, to date, no study has compared the efficacy and safety of both treatments. The objective of this study was to compare the efficacy and safety of patients with advanced HCC who received either TACE-S or TACE-A.  193 patients with advanced HCC were included in the study between June 2015 and December 2019. Propensity score matching (PSM) analysis was used in the study to reduce selection bias. P=0.703, P=0.514). TACE-A did not increase the mortality risk compared with TACE-S in the first 12 months  or after the 12-month mark . Similarly, TACE-A did not increase the tumor recurrence risk relative to TACE-S in the first 12 months  or after the 12-month mark . The subgroups analysis showed that TACE-A did not increase mortality risk or tumor recurrence risk relative to TACE-S. After PSM, similar results were presented. The III and IV stage adverse events in the TACE-A group were similar to those in the TACE-S group before PSM.  Before PSM, the median overall survival (mOS) and median progression-free survival (mPFS) of patients treated with TACE-S were not significantly longer than in patients treated with TACE-A ( Patients with advanced hepatocellular carcinoma could get similar survival benefits from treatment with either transarterial chemoembolization plus apatinib or transarterial chemoembolization plus sorafenib. Hepatocellular carcinoma (HCC) is one of the most common cancers in the world . At presApatinib is an antiangiogenic drug that targets the vascular endothelial growth factor receptor-2 (VEGFR-2) and exerts similar antitumor effects to sorafenib.Apatinib can target VEGF-R-2, inhibit the activation of VEGFR-2, and then block its downstream signal by specifically competing for adenosine triphosphate binding site in the cell. It can also inhibit the proliferation migration and the tube formation of human umbilical vein endothelial cells, blocking the generation of aortic rings , 13. In Research has shown that patients treated with TACE-S or TACE-A had better survival benefits than patients treated with monotherapy (TACE or sorafenib) , 19, 20.We retrospectively reviewed the medical records of 409 consecutive patients with advanced HCC who received TACE-S or TACE-A from June 2015 to December 2019 in our institution. Before the initial TACE procedure, TACE-S was firstly recommended. For patients who refused to receive sorafenib, TACE-A was recommended.9/L. The exclusion criteria were as follows: (1) patients received TACE or sorafenib or apatinib before they were included in the study; (2) patients with tumors that were metastasized from other organs; and (3) patients with diffuse tumors which could not be evaluated (The inclusion criteria of the study were as follows: (1) patients were diagnosed as advanced HCC based on the EASL guideline; (2) the Child-Pugh score of patients was A or B; (3) the Eastern Cooperative Oncology Group of patients was 0, 1, or 2; and (4) the platelet counts of patients were higher than 60 × 10valuated . This stTACE procedures were performed by three experienced radiologists with a minimum of 10 years of experience in interventional therapy. A 5 F catheter  was inserted into the hepatic artery. Then, a 3F micro-catheter  was used to insert the tumor-feeding arteries. Subsequently, lipiodol  was mixed with doxorubicin hydrochloride to create an emulsion at the ratio of 1 mL/2 mg. Depending on the size of the tumor and liver function, 5–20 mL emulsion was injected slowly into the tumor-feeding arteries until stasis of the feeding arteries of the tumor. If it was necessary, supplement embolization was performed using gelatin sponge particles .In the TACE-S group, sorafenib was orally and continuously administered 400 mg twice daily when TACE was performed. In the TACE-A group, apatinib was orally taken 3–5 days after each TACE procedure at the starting dose of 500 mg/day. The dose adjustments were based on the tolerance to the drug. The grading of adverse events associated with apatinib and sorafenib was conducted according to the National Cancer Institute Common Terminology Criteria for Adverse Events (version 4.0). In the case of grade 3 or 4 apatinib- or sorafenib-related adverse events, the dose of apatinib was modified to 250 mg/day and the dose of sorafenib was adjusted to 400 mg once daily until the adverse effects were alleviated.The primary endpoint of the study was overall survival (OS), which was defined as the time from the first TACE to patients' death or the end of the study. The secondary endpoints were progression-free survival (PFS) and disease control rate (DCR) 3 months after the initial TACE. The PFS was defined as the time from the initial TACE to the progression of the tumor, the time of patients' death, or the time of the end of the study. The DCR was defined as the proportion of patients with complete response (CR), partial response (PR), and stable response (SD) in patients with TACE-S or TACE-A based on the mRECIST criteria .All patients who were included in the study were followed up. In the study, patients were required to receive CT or MRI and laboratory tests every 4–6 weeks after the initial TACE for 6 months. After 6 months, patients were required to receive CT or MRI and laboratory tests every 2-3 months. The images of patients were reviewed by two radiologists (one with 24 years of radiographic experience and one with 20 years of radiographic experience) who were blinded to the treatment of the patients. If the results of their evaluation were different, another radiologist (with 31 years of radiographic experience) evaluated the results and gave the final results. When the CT or MRI image showed that the tumor remained or the tumor progressed, another TACE was recommended to these patients. The follow-up end time of the study was July 30, 2020.t-test or Mann–Whitney U test. Categorical variables were compared by the Chi-square test or Fisher's test between the two groups. Kaplan–Meier was used to plot the survival curves and the survival benefits in both groups and compared by log-rank. Adjusted Cox regression risk model was used to predict the potential factors which might influence the OS and PFS. Propensity score matching (PSM) was used to reduce selection bias. All characteristics were included in PSM analysis, and 1 : 2 ratio matching with an optimal caliper of 0.2 without replacement generated 139 patients (49 patients with TACE-S and 88 patients with TACE-A). The Landmark method was used in the study because it determines survival based on response status assessed at each specific time point. Because the Landmark is recommended in oncology guidelines for TACE-S/TACE-A analysis, a 12-month Landmark for OS and PFS was used in the study before and after PSM. All statistical analysis was conducted by SPSS 24.0  and R 3.6.2.Continuous variables were compared by independent sample In this study, a total of 193 patients with advanced HCC who received TACE-S or TACE-A were included in the analysis. Among them, 54 patients received TACE-S and 139 patients received TACE-A. The median time of follow-up was 11.5 months  in the TACE-S group and 11.5 months  in the TACE-A group. In the TACE-S group, 43 patients of the 54 died and 120 patients of 139 patients died in the TACE-A group during the follow-up .P=0.703). The mPFS was 5.7 months (95%CI: 5.0–6.4 months) in the TACE-S group and 5 months (95%CI: 3.8–6.2 months) in the TACE-A group (P=0.514) (P=0.311). After PSM, the mOS and mPFS were 12.2 months (95%CI: 8.9–15.5 months) and 5.4 months (95%CI: 4.4–6.4 months) in the TACE-S group and 11.9 months (95%CI: 10.2–13.6 months), and 5.1 months (95%CI: 4.0–6.2 months) in the TACE-A group (P=0.672 and P=0.808) (P=0.726).Before PSM, the mOS was 12.4 months (95%CI: 10.7–14.1 months) in the TACE-S group and 11.5 months (95%CI: 8.8–14.2 months) in the TACE-A group (P=0.514) . The DCRP=0.808) . The DCRP=0.329) or tumor recurrence risk  compared to TACE-S in the previous 12 months. After 12 months, TACE-A also did not increase the mortality risk  or tumor recurrence risk  compared to TACE-S (P=0.904) or tumor recurrence risk  compared to TACE-S in the previous 12 months. After 12 months, TACE-A also did not increase the mortality risk  or tumor recurrence risk  compared to TACE-S (P=0.620) or tumor recurrence risk  compared to TACE-S before PSM (P=0.744) or tumor recurrence risk  compared to TACE-S. After 12 months, TACE-A did not increase the mortality risk  but reduced the tumor recurrence risk  compared to TACE-S  or tumor recurrence risk  compared to TACE-S in the patients with Child-Pugh A and B scores. Similarly, TACE-A did not increase the mortality risk  or tumor recurrence risk  compared to TACE-S in the patients with extrahepatic metastases and without extrahepatic metastases. Again, TACE-A also did not increase mortality risk  or tumor recurrence risk  compared with TACE-S in the patients with portal invasion and without portal invasion before PSM , abdominal pain (P=0.886), nausea (P=0.443), vomiting (P=0.235), anorexia (P=0.692), diarrhea (P=0.749), hypertension (P=0.085), fatigue (P=0.343), hand-foot reaction (P=0.715), gastrointestinal hemorrhage (P > 0.999), headache (P=0.921), and proteinuria (P=0.892) in the TACE-S group and TACE-A group. Similarly, there was no statistically significant difference of relative adverse events (grades ≥3) between the two groups   .Although the SHARP and ORIENTAL clinical trials showed that patients with advanced HCC who received sorafenib had better survival benefits than a placebo group, the response rate of patients who received sorafenib was still low , 22–24. Previous studies have shown that the mOS of advanced HCC patients with TACE-S ranged from 11.2 months to 17.5 months, and the median time to progression (mTTP) or mPFS ranged from 4.3 months to 7.6 months , 25–28. In the study, the Landmark was used to mitigate guarantee-time bias, and a 12-month period was used , 32. TheIn the study, the adverse events related to TACE or apatinib or sorafenib were evaluated. The III and IV grades of adverse events in the TACE-A treatment group were not higher than the TACE-S treatment group. And after reducing the dose administration of apatinib, the adverse events of patients with TACE-A and TACE-S were relieved. The results of this study showed that apatinib is a well-tolerated treatment option with an acceptable safety profile for patients with advanced HCC compared with sorafenib.Some limitations existed in the study. Firstly, the study was a retrospective study, which led to the existence of selection bias. However, the PSM was used in the study to reduce selection bias. Secondly, the number of patients enrolled in the study was small, especially the sample of patients with TACE-S, which might influence the accuracy of the conclusion. Finally, the selection of drugs might be influenced by the patients' willingness, which might result in some patients receiving suboptimal treatments. However, until now, there have been no criteria indicating which kind of advanced HCC patients are suitable for sorafenib or apatinib. Consequently, more studies are needed to confirm the results of the study.The study showed that patients with advanced HCC who received TACE-A had comparable survival benefits to patients who received TACE-S. TACE-A might be a good choice for patients who were unwilling to receive TACE-S.


Mytilus galloprovincialis, elements of the three peptide-receptor systems are highly abundant in the mantle an innate immune barrier tissue. Exposure of M. galloprovincialis to lipopolysaccharide or a marine pathogenic bacterium, Vibrio harveyi, provoked significant modifications in the expression of genes of the peptide precursor and receptors of the AST-C-like system in the mantle suggesting involvement in the immune response. Overall, our study reveals that homologues of the arthropod AST-systems in molluscs are potentially more complex due to the greater number of putative mature peptides and receptor genes. In bivalves they have a broad and varying tissue distribution and abundance, and the elements of the AST-C-like family may have a putative function in the immune response.The allatostatins (ASTs), AST-A, AST-B and AST-C, have mainly been investigated in insects. They are a large group of small pleotropic alloregulatory neuropeptides that are unrelated in sequence and activate receptors of the rhodopsin G-protein coupled receptor family (GPCRs). The characteristics and functions of the homologue systems in the molluscs , the second most diverse group of protostomes after the arthropods, and of high interest for evolutionary studies due to their less rearranged genomes remains to be explored. In the present study their evolution is deciphered in molluscs and putative functions assigned in bivalves through meta-analysis of transcriptomes and experiments. Homologues of the three arthropod AST-type peptide precursors were identified in molluscs and produce a larger number of mature peptides than in insects. The number of putative receptors were also distinct across mollusc species due to lineage and species-specific duplications. Our evolutionary analysis of the receptors identified for the first time in a mollusc, the cephalopod, GALR-like genes, which challenges the accepted paradigm that AST-AR/buccalin-Rs are the orthologues of vertebrate GALRs in protostomes. Tissue transcriptomes revealed the peptides, and their putative receptors have a widespread distribution in bivalves and in the bivalve  Molluscs are the second most diverse animal group after the insects and belong to the speciose Lophotrochozoan clade. Their success is linked to their adaptation to a wide variety of habitats, and they are found from the abysses of the sea to mud flats and even as parasites dwelling in other animals. Unlike the more popular protostome models of the nematodes and insects that have substantial genome rearrangements and gene loss –4, the mThe allatostatins (AST) are a diverse group of small neuropeptides that have low sequence conservation but have overlapping functions as insect allatoregulatory peptides . Three AThe members of each AST family derive from distinct precursor proteins that are assumed to undergo proteolytic cleavage to generate multiple peptides with a similar structure and sequence. The exception is the precursor of AST-C which encodes a single peptide. AST peptides bind and activate members of the rhodopsin G-protein coupled receptor (GPCR) superfamily. AST-A and AST-C activate receptors of the rhodopsin-gamma GPCR cluster while AST-B activates receptors of the rhodopsin-beta GPCR cluster –17. SequDiploptera punctata (Periplaneta americana and Diploptera punctata) to 4 to 5 in Diptera (Drosophila melanogaster and Anopheles gambiae)   their presence in the mantle has been linked to a role in shell formation  , 32–35. gambiae) . A singlgambiae) , 38. In  feeding , 24. Curomerata)  and in tormation .D. melanogaster the AST-B precursor gives rise to 5 peptides but in Rhodnius prolixus 12 peptides are generated  (Gryllus bimaculatus) (Bombyx mori) (D. melanogaster) (Leucophaea maderae) (Locusta migratoria) (Scylla paramamosain)  . The allculatus) . Subsequyx mori) , 43, theogaster) , feedingmaderae) , reproduratoria)  and the amosain) . In inseamosain) , 47, 48.amosain) . The ASTamosain) , 50, 51 amosain) , 16.Manduca sexta)   and unlia sexta) , 54. Thea sexta) , 55, 56.a sexta) –62. In Dimmunity . Receptoimmunity , 63, 64.M. galloprovincialis) exposed to an immune challenge with bacteria lipopolysaccharide (LPS) and heat inactivated Vibrio harveyi, a common pathogenic bacteria of the marine environment, was established  and AST-CRs . Searches were performed against molecular data available in NCBI (https://www.ncbi.nlm.nih.gov) using blastp in the species-specific sub-datasets available for five bivalves: two members of the Mytilidae family, M. galloprovincialis (taxid:29158) and Mytilus coruscus (taxid:42192), two members of the Ostreidae family, Crassostrea gigas (taxid:29159) and Crassostrea virginica (taxid:6565) and one member of the Pectinide family, the scallop Mizuhopecten yessoensis (taxid:6573); three gastropods, Aplysia californica (taxid:6500) of the Aplysidae family, Biomphalaria glabrata (taxid:6526) of the Planorbidae family and Lottia gigantea (taxid:225164) of the Lottidae family; and also one cephalopod the octopus Octopus bimaculoides (taxid:37653).Searches for the homologues of the insect AST-GPCRs  were performed in molecular data available for nine representative species of the Mollusca phylum using the deduced protein sequences of the previously identified s AST-AR ] and D. https://www.ncbi.nlm.nih.gov/genome/) were also procured using the tblastn algorithm with the M. galloprovincialis deduced protein sequences of AST-like receptors as the query (Capitella teleta GALR-like (ELU13887.1) for other protostome orthologues.To increase the number of receptor sequences from the phylum Mollusca, selected genomes of representatives of different orders available from the NCBI Genomes database (Lingula anatina (taxid:7574), two annelids Capitella teleta (taxid:283909) and Platynereis dumerilii (taxid:6359) and in three arthropods of the Ecdysozoans namely, the diptera Anopheles gambiae (taxid:7165), the coleoptera Tribolium castaneum (taxid:7070) and the branchiopod Daphnia pulex (taxid:6669). Sequences for the homologue receptors in the vertebrates, human , spotted gar  and for the invertebrate deuterostome, the amphioxus  were retrieved for comparison with the Lophotrochozoan and Ecdysozoan sequences. Sequence hits with a cut off < e-30 were retrieved, and their identity was confirmed by searching against the D. melanogaster (taxid:7227) and H. sapiens (taxid:9606) databases.For comparative analysis, sequence searches were also performed in other Lophotrochozoan phyla namely, the brachiopod M galloprovincialis, SRP 063654)  to identify the corresponding gene transcripts. Putative receptors were retrieved based on their sequence similarity (cut off < e-30) with M. galloprovincialis and D. melanogaster homologues or using transcriptome annotations. All sequences retrieved were translated using the Expasy translate tool (https://web.expasy.org/translate/) and their identity confirmed as described above.Searches were also performed in mantle transcriptomes available from the Mediterranean mussel ( 063654)  and the M. galloprovincialis and M. coruscus orthologues of the insect AST-B/MIP and AST-C peptide precursors were initially obtained by exploring the species-specific mantle transcriptome annotation available “in house”. The deduced protein sequence of MIP-like and AST-C-like were used to identify the corresponding coding genes and additional gene isoforms (https://web.expasy.org/translate/) and the localization of the predicted mature peptides was manually deduced by a) the identification of monobasic, dibasic, or tribasic consensus cleavage sites  and b) the identification of the conserved mature peptide motifs such as the two conserved tryptophan (W) residues (W(X)W-amide) in AST-B/MIPs and the two conserved cysteine (C) residues in AST-Cs. The mature peptides and the full peptide precursors were procured in other molluscans  using a similar strategy. Homologues from other representatives of the lophotrochozoan clade such as one brachiopod and two annelids were also characterized for comparisons  and L. oculeatus (XP_006629714) cholecystokinin receptor type A (CCKAR) branch. To build the phylogenetic trees for the AST-Rhodopsin γ family GPCRs the sequences of the metazoan KISSRs and GALRs were also included as they are suggested to have evolved from the same ancestral gene as protostome Buccalin-R/AST-AR  and als6637109) , 16, 79.http://www.nrbsc.org/gfx/genedoc/). The mature peptide alignments were established using Clustal W . Receptor structures were predicted in Protter (http://wlab.ethz.ch/protter) using the default settings and the outputs annotated in Inkscape to highlight the conserved aa positions across species. The receptor signal peptide was predicted using the SignalP-5.0 Server  and SuperContig CAPTEscaffold_45 (scaffold_45), respectively and H. sapiens GALRs and D. melanogaster AST-ARs (DAR1 and DAR2) were characterized to infer potential ancestral origin and to support the identification of the new protostome sister clade of the deuterostome GALRs. Ten genes upstream and downstream of the C. teleta gene loci were collected and they were used to search for gene homologues in the H. sapiens GALR1 (chromosome 18), GALR2 (chromosome 17), GALR3 (chromosome 22) and KISSR (chromosome 19) and in D. melanogaster DAR1 (chromosome X) and DAR2 (chromosome 3R) genome regions. The BioMart tool available from Ensembl was used to compare the C. teleta and D. melanogaster genome regions. The deduced C. teleta buccalin-R and GALR-like neighbouring protein sequences were searched against the species-specific H. sapiens (taxid:9606) and D. melanogaster (taxid:7227) datasets available from NCBI (https://www.ncbi.nlm.nih.gov). The gene loci of the top protein hits  were retrieved based on the genome assemblies available from NCBI . The O. bimaculoides GALR-like genome region on SuperContig KQ419625 was also analysed and the five neighbouring genes were retrieved and searched against C. teleta and H. sapiens genomes.The neighbouring gene environments of the annelid M. galloprovincialis) were obtained from a local producer in the Ria Formosa . For the experimental immune challenge mussels  were transported live to the Centre of Marine Sciences (CCMAR) where they were cleaned and acclimatized for a week in 5 litres of aerated seawater (SW) prior to the immune challenge at 20 - 22°C. Animals were fed daily with a mixture of a commercial dried microalgae diet . For tissue sampling animals were opened by cutting the adductor muscle and the mantle edge from the region most distal to the umbo (referred to as the posterior region) was dissected out and snap frozen in dry ice and stored at -80°C for RNA extraction. For tissue distribution, cDNA samples (n = 3 for each tissue) from gills, digestive gland, mantle edge and haemolymph available in the lab were used.Mediterranean mussels  was grown in TSB medium supplemented with 1% NaCl and the number of cfu/ml was determined on TSA/1% NaCl agar plates. For the bacterial challenge 5 x 107 cfu/ml of the heat inactivated bacteria suspended in 1 L of sterile seawater was used. The V. harveyi bacteria was heat inactivated by boiling the culture for 2 hours.Mussels were exposed to heat-inactivated V. harveyi (5 x 107 cfu/litre) and then transferred to new tanks containing clean filtered seawater. Specimens (n = 6 per timepoint) from control and challenged tanks were sampled (as outlined above) at 0, 6, 12, 24 and 36h post exposure. Animals were not fed during the experiment and no mortality was observed.For the challenge experiments mussels (n = 80) were randomly distributed in triplicate tanks. The experiments were performed in 2 L plastic tanks containing 1 L of filtered seawater (0.45 μm) that was collected from their natural environment. Each of the control (3 tanks) and exposed (3 tanks) tanks contained 12 - 13 individuals . The seawater in the experimental tanks was constantly aerated with aquarium air-pumps and the temperature was 20 - 22°C, pH was 8.1 ± 0.1 and salinity 37 ppt and the experiments were conducted under natural photoperiod for March 2021 in the Algarve. Control mussels were maintained in seawater and transferred after 15 h to new tanks and the immune challenged mussels were exposed for 15 h to heat-inactivated Total RNA (tRNA) from control and immune challenged mantle edge was extracted using an E.Z.N.A kit  and DNase treatment was performed after elution using a Precision DNase kit  according to the manufacturers protocol. For extraction, collected tissues were defrosted in the lysis buffer and homogenized by mechanical disruption with two iron beads (5 mm) using a Tissue lyser II Qiagen and 1 cycle of 3 min at room temperature.18S rRNA using specific primers  was denatured at 65°C for 5 min and quenched for 5 min on ice. Reactions were carried out for a 20 μl final volume with 10 ng of pd(N)6 random hexamers , 2 mM dNTPs , 100 U of RevertAid Reverse Transcriptase and 8 U Ribolock RNAse inhibitor (ThermoScientific). Reaction conditions were: 10 min, 20 °C; 60 min, 42 °C; 5 min , 70 °C. The quality of the cDNA produced was assessed by amplification of mussel ribosomal subunit  primers Table 1 M. galloprovincialis, M. coruscus, C. gigas and M. yessoensis were retrieved and analysed. Public transcriptome data available for control tissues including the gills, muscle, mantle, digestive gland/hepatopancreas, haemocytes and nerve ganglia were searched using the species-specific nucleotide sequences identified for the peptide precursors and receptors. Searches were carried out using Blastn and the corresponding sequence read archive  for each of the species analysed  a major component of the outer membrane of Gram-negative bacteria. Candidate transcripts were identified by exploring available in-house DEG data  for the mantle edge transcriptomes of control M. galloprovincialis (injected with 1x PBS) and LPS exposed M. galloprovincialis  from samples collected in the context of another study.The involvement of the bivalve homologues of the arthropod ASTs and receptors in the immune response was initially assessed using mantle edge transcriptomes of M. galloprovincialis members was assessed using cDNA (n = 3) from normal tissues  and from the mantle edge of control and exposed specimens to heat-inactivated pathogen, V. harveyi, at 0 (n = 6), 6 (n = 6), 12 (n = 6), 24 (n = 6) and 36 h (n = 6) post exposure. Specific primers for the M. galloprovincialis AST-C-like peptide precursors and for four AST-CR-like were designed and the PCR products were sequenced, and their identity confirmed . The activation of the immune response in M. galloprovincialis was confirmed by determining the expression of three humoral factors that have previously been shown in bivalves to respond to Vibrio spp., Toll-receptor TLRa  in cDNA of the mantle edge of control (n = 3) and exposed (n = 3) specimens from 0, 6 and 12 h post exposure  and 18s ribosomal subunit (18S) were used as reference genes . QRT-PCR analysis was performed in duplicate reactions (< 5% variation between replicates) using a CFX Connect™ Real-Time PCR Detection System for 96-well microplates (Bio-Rad). Cycling conditions were 95°C, 30 sec; 44 cycles of 95°C, 5 sec; the most appropriate primer annealing temperature, 10 sec (2 (coefficient of determination) were established  using SsoFast EvaGreen Supermix  in a 10 µl final reaction volume containing 200 nM of forward and reverse gene specific primers Table 1 www.graphpad.com).Results are presented as the mean ± SEM. Statistical differences were detected using One-Way ANOVA for the tissue distribution and for gene expression between the control and immune challenged mussels with Two-Way ANOVA using a Sidak’s multiple comparison test. Analysis was executed using GraphPad Prism version 8.0 for Mac OS X  actions on JH biosynthesis from the insects CA gland. In molluscs no equivalent organ has been described and JH is specific to insects. In molluscs the sequence orthologues of the arthropod AST-A are known as buccalin and in annelids the orthologues of the arthropod AST-B/MIP are known as MIP. For the lophotrochozoan AST-Cs no nomenclature has yet been proposed. Throughout the manuscript we use the existing lophotrochozoan nomenclature and have named AST-C as AST-C-like Table 2.Sequence homologues of the three arthropod AST peptide precursors and receptors were found in the 27 mollusc species included in the study and the number of peptide precursor genes and predicted mature peptides and putative receptor genes varied across the species (Conus ventricosus (CM031604.1 and CM031615.1) and Pomacea maculata (XP_025108540.1 and XP_025087986.1) where two putative genes were found. The number of buccalin-R was more variable and while most species possessed a single buccalin-R gene, other species genomes contained two receptor genes , Pinctada imbricata (Pteriidae family) and in Tegillarca granosa (Arcidae family) at least 8, 8 and 10 putative mature peptides respectively, were encoded, and the full-length precursors still remain to be isolated. No buccalin precursor genes were retrieved from the bivalves Sinonovacula constricta and Ruditapes philippinarum genomes.The buccalin precursors encoded for a different number of peptides. In the Mytilidae family 7 mature peptides were predicted in A. californica (Aplysiidae family) contained the largest number of predicted peptides (29 mature peptides) and the Gigantopelta aegis (Peltospiridae family) precursor was the second most rich. In the cephalopod O. bimaculoides and most cephalopod genomes explored our searches failed to identify the buccalin precursor except in the Nautilus pompilius (Nautilidae family) where 29 peptides were predicted  but in other taxa at least 6 peptides existed  were found. In contrast, in other molluscs receptor number was variable and 5 putative AST-CR-likes were retrieved from bivalves of the Mytilidae family and four were found in the gastropod C. unifasciata (Arionidae family). The other representatives of the diverse Mollusca classes possessed 1 to 2 AST-CR-like . An orthologue sequence was identified in the genome of M. galloprovincialis (UYJE01007806.1) when the M. coruscus AST-C-like sequence was used for searches.In Mollusca a single AST-C-like peptide precursor that encodes a single mature peptide was found in all the species analysed  and P. dumelii (AKQ63078) and with the deuterostome GALRs  were identified in molluscs and other lophotrochozoans and the clustering of the retrieved bivalve sequences suggested that there were three MIP-R subtypes Figure 3M. galloprovincialis and M. coruscus. In addition, the clustering of one of the Ostreidae duplicate receptors from C. gigas, C. virginica and M. hongkongensis in distinct branches from the other lophotrochozoan receptors suggested that the two gene copies were under different evolutionary pressure   Figure 4A. californica buccalin precursor encoded the largest number of predicted peptides (29 mature peptides) and 25 were L-amine and the remaining 3 were C-terminal I-amide and produced 17 different peptides  but the B. glabrata precursor only encoded I-amide peptides   with Mga_d, e and f.In gastropods, the peptides Figure 4M. coruscus to 12 in M. galloprovincialis but in the gastropod C. unifasciata two MIP precursors existed and they encoded for different peptides  were identical and the peptides overall, shared 22 to 62% aa identity  and it encoded putative mature peptides of different sizes, and the majority were slightly longer (12 aa) than most molluscan peptides due to their extended N-terminal region W, where x represents any aa). In D. melanogaster there were five AST-B/MIP peptides that contained two conserved W residues separated by 6 aa (W(x6)W). The 12 MIP mature peptides in the bivalve M. galloprovincialis shared 38 to 78% aa sequence identity with the D. melanogaster orthologues.The MIP precursors in common with the mollusc buccalin precursor have the potential to generate multiple peptides by proteolysis of a single polypeptide precursors and the highest peptide numbers were identified in the gastropods  residues that form a disulphide bond in the peptide (D. melanogaster AST-C (25 - 33%) than with AST-CC (13 - 17%).A single AST-C-like peptide was encoded by the AST-C precursor and in the molluscs analysed, the peptide was 13-15 aa in length . The three M. galloprovincialis MIP-Rs were only 25 - 29% identical suggesting that they may have different functions. The M. galloprovincialis type I receptor was most similar to D. melanogaster AST-BR (35%), while the other paralogues type II and type III only shared 20% identity with D. melanogaster AST-BR. The amino acid sequence of the five M. galloprovincialis AST-CR-like shared between 37 to 80% identity and VDI08560.1 and VDI60978.1 (that arose due to a recent gene duplication) shared the highest aa sequence identity. The M. galloprovincialis and M. coruscus AST-CR-like shared 36 – 94% aa identity and 27 to 34% aa identity with the two D. melanogaster receptors.The deduced protein sequence of C. teleta and O. bimaculoides, which have the two receptor types was characterized (O. bimaculoides GALR-like gene mapped to a short genome fragment (SuperContig KQ419625) and only 5 gene neighbours were mapped, and searches against C. teleta and the human genome regions failed to identify gene homologues in the neighbourhood of the GALR-like and GALRs genes.Previously, our studies on the characterization of the Buccalin-R/AST-ARs and the sequence homologues of the vertebrate KISSR and GALRs in Arthropods and Mollusca suggested that they were most closely related to the metazoan KISSRs , 37. Howcterized Figure 6C. teleta the GALR-like homologue mapped to scaffold_148 and gene homologues of human CEP131 and HID1 located near the loci of human GALR2 in chromosome 17 were identified , GALR2, GALR3 (chromosome 22) or KISS1R (chromosome 19) genome regions. Comparison of the gene environment of human KISSR and AST-ARs in D. melanogaster (chromosomes 3R and X) and A. gambiae (chromosome 2R) using the conserved neighbouring genes previously identified, PTBP1, EVIL5L, DOT1L and ODF3L2  of the peptides and receptors suggests that they may have different functions , and were far more abundant in this tissue than in the mantle , was significantly down-regulated 12h post challenge  were detectable in the mantle  and AST-CR-like VDI53419.1 was significantly more expressed (p < 0.01) in the gills than in other tissues. AST-CR-like VDI53419.1 was most abundant and its expression in tissue was approximately 10-fold higher than the other receptor gene VDI15122.1 and the gene encoding the peptide precursor  analysis of e mantle Figure 8M. galloprovincialis to heat-killed V. harveyi was assessed by measuring by qRT-PCR the change in expression of innate immune-related genes. TLRa was significantly up-regulated 6h (p < 0.001) and 12 h (p < 0.05) after an immune challenge and LYG1 was significantly down-regulated at 6 h (p < 0.05) and complement-like C3-like was not significantly changed (AST-C-like peptide precursor and receptor expression was modified after exposure to heat-killed V. harveyi (AST-C-like precursor was significantly up-regulated 6 h (p < 0.05), 12 h (p < 0.0001) and 24 h (p <0.01) after an immune challenge. AST-CR-like VDI15122.1 was down-regulated at 6 h (p < 0.01), 12 h (p < 0.001) and 24 h (p < 0.0001) and AST-CR-like VDI53419.1 was down-regulated at 6 h (p < 0.0001) and 12 h (p < 0.01) after exposure to heat-killed V. harveyi  a group of ecologically and commercially important species. In molluscs, homologues of the three arthropod peptide groups  and their corresponding receptors were identified. The sequence similarity between the mollusc and insect peptides and receptors was low but aa motifs involved in peptide and receptor structure were well conserved. In molluscs the peptide precursor and receptors evolved D. melanogaster and other insects. In common with the insects and other arthropods AST-A and AST-B/MIP, the buccalin and MIP mature peptides in Mollusca were encoded by long protein precursors containing multiple mature peptides with distinct amino acid sequences but well conserved motifs important for bioactivity  are authentic as they both encode C-terminal L-amide peptides  muscle of the gastropod  arousal . In arth V-amide  was excl V-amide , 20, 25.peptides  as well nd GALRs , 37, 38.nd GALRs , 37 but 6/7)W-amide sequence in Arthropoda AST-B/MIP peptides (4-7)W-amide in Mollusca MIPs. Gastropod MIP precursors produced the largest variety of peptides and in L. gigantea 25 peptides were predicted, which was 5 times higher than in the insect D. melanogaster. Like the insects, D. melanogaster and T. castaneum a single AST-BR/MIPR was identified in most gastropods and in cephalopods. In contrast, bivalves possessed three receptor type genes that arose prior to the divergence of the molluscs and arthropods by gene duplication. In insects, AST-B/MIP share the same receptor as insect sex peptide, a.k.a. accessory gland peptide, which in D. melanogaster is 36 aa in length. The single gastropod A. californica MIP-R has been deorphanized but the species MIP peptides , but a larger number of putative AST-CR-like exist compared to arthropods. In molluscs the receptor number was generally conserved apart from in the bivalves and specifically the Mytilidae family where lineage and species-specific events caused expansion of the AST-CR-like (type I). The oysters possessed the protostome AST-CR-like type I and an additional AST-CR-like type II that according to the phylogeny diverged from other lophotrochozoan receptors. Genome analysis of the lophotrochozoans revealed that AST-CR-like type II are tandem duplicates with the AST-CR-like type I gene suggesting that after gene duplication in the ancestral Ostreoidea they considerably diverged.In molluscs the AST-C-like peptide precursor generated a single peptide as observed in arthropods. The AST-C mature peptides were characterised by two conserved cysteine motifs in arthropods , 54, 92,The identification in molluscs of homologues of the arthropod AST peptide precursors with the potential to produce multiple mature peptides with distinct sequences and of AST-Rs that emerged from lineage/species-specific expansions in molluscs suggests that the diversity and complexity of this neuropeptide system is higher than in arthropods. The functional role of the arthropod AST-like system in the molluscs and other Lophotrochozoans remains to be studied but its diversity suggests it may be pluripotent. In the future, deorphanization of the multiple receptors within the Mollusca with the corresponding peptides will help to understand the peptide-receptor interactions and provide a route towards the establishment of function.The lack of sequence conservation and distinct peptide precursor organization of the three protostome AST-like peptide families  is indicative of their different evolutionary origins. This contrasts with the common evolutionary origin proposed for cognate receptor families. The presence of both peptide precursors and receptors in arthropods and molluscs suggested that they emerged prior to the ecdysozoans-lophotrochozoan divergence and shared a common origin and functional conservation with homologue systems in vertebrates. The protostome AST-A/Buccalin systems are homologues of the highly studied deuterostome GALR/KISS systems and the AST-C/AST-C-like system in protostomes is suggested to be the counterpart of the vertebrate SST-system –65, 95. AST-AR and GALRs in the annelid (C. teleta), insect (D. melanogaster) and vertebrate (H. sapiens) revealed orthologue genes. The GALR-like genome region in C. teleta possessed genes shared in the genome region flanking arthropod AST-AR and human GALR2 suggesting that the protostome GALR-like and vertebrate GALR genome regions are related and that the ancestral genes of metazoan GALR and protostome AST-ARs may have emerged from a common genome region in the early bilaterian genome prior to the protostome and deuterostome divergence. The Buccalin-R genome regions in C. teleta only shared genes with the equivalent AST-AR genome regions in D. melanogaster and no orthologues were found in human GALR or KISSR genome regions. Nonetheless, the evolutionary scenario that gave rise to the protostome ancestral AST-ARs, metazoan KISSR and GALR genes is difficult to resolve since protostomes and deuterostomes are suggested to have diverged approximately 535 million years ago  amongst others , 99, 100Diploptera punctate ASTs were suggested to be important regulatory peptides of the immune response  were up-regulated in the haemocytes 6 hours after infection with the white spot syndrome virus  pathway  were distinct across mollusc species, and this suggests that the AST families may be as complex as the neuropeptide system described in insects and other arthropods.Phylogenetic analysis confirmed that receptors are specific to protostomes and emerged early in evolution prior to the Lophotrochozoa and Ecdysozoa divergence. In a previous study  we propoM. galloprovincialis significantly changed the expression of the AST-C-like peptide and receptor genes supporting its role in immunity and hinting at conservation of this function across protostomes. Overall, our study provides for the first time a comparison of the homologues of the three arthropod AST-systems across different molluscs and contributes to a better understanding of their evolution and function in protostomes.Peptide precursors that encode for multiple mature peptides that diverge in sequence and the existence of lineage and species-specific duplicate receptors in molluscs suggests that the function of the Buccalin, MIP and AST-C-like systems may be distinct across species and that adaption to different environments may have affected gene evolution. Meta-analysis of tissue transcriptomes revealed that the Buccalin, MIP and AST-C-like families have a broad tissue distribution and varying abundance in several different bivalves, indicating it is an omniscient regulatory factor of the molluscan neuropeptide repertoire. The administration of an immune challenge to The sequence, accession sequence numbers and datasets analyzed in this study are included in the Figures and also available in article/JC and DMP planned and supervised the study. JC and JI performed the bioinformatic and comparative analysis. ZL and MP performed the experimental challenge, transcriptome analysis and expression studies. JC and DMP analysed the results and wrote the manuscript. All authors contributed to the article and approved the submitted version.This study was funded by the Portuguese Foundation for Science and Technology (FCT) through project UIDB/04326/2020. ZL was supported by a PhD scholarship from the China Scholarship Council. MP was supported by a PhD scholarship from Shanghai Ocean University (China).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Background: Individuals with insulin resistance (IR) have a high risk of diabetes or metabolic syndrome, and they are more likely to have depression. Furthermore, IR by itself is a major cardiovascular risk factor in healthy persons. Thus, we aimed to investigate IR in association with thyroid function, psychoemotional state, and cardiovascular risk factors among 45–84-year-old citizens of Palanga. Methods: A randomized epidemiological study was performed with 850 subjects. All participants were evaluated for sociodemographic, clinical, and cardiovascular risk factors and biochemical analysis. IR was evaluated by the homeostasis model assessment of IR (HOMA-IR). Results: All study participants were stratified into groups without IR (HOMA-IR ≤ 2.7) and with IR (HOMA-IR > 2.7). The analysis of parameters between the two study groups showed some statistically significant relationships between IR and cardiovascular risk factors. The predictable accuracy was presented using receiver performance characteristic curves for HOMA-IR scores in women and men separately. If the HOMA-IR score is higher than 3.45, individuals are significantly more likely to have type 2 diabetes mellitus (T2DM). Conclusions: An increase of fasting glucose and more frequent incidence of metabolic syndrome, diabetes, and cardiovascular diseases in subjects with IR are associated with the prevalence of cardiovascular risk factors. There was no significant association between thyroid function and HOMA-IR. HOMA-IR cut-offs could predict the presence of T2DM. Insulin resistance (IR) occurs when insulin no longer effectively stimulates glucose uptake in metabolic tissues. The inability of metabolic tissues to take up glucose results in hyperglycemia and hyperinsulinemia, both hallmark symptoms of IR. Most individuals with IR go undiagnosed, and the condition can continue for 10–12 years, which can be especially damaging as IR is an independent risk factor for obesity, cardiovascular disease, arterial hypertension (AH), and type 2 diabetes mellitus (T2DM). This time span is an important intervention window where it is possible to prevent and reverse progression towards metabolic disease and T2DM .In daily clinical practice, the determination of IR indices is based on measurements of fasting glucose and insulin concentrations. Homoeostatic model assessment of IR (HOMA-IR) is the most commonly used indirect indices for defining this condition in the “steady state” ,5. ThyroThe etiology of metabolic disorders is complex and multifactorial. The key causes are obesity, physical inactivity, sedentary lifestyle, and aging population. Recently, the role of psychological disorders on metabolic parameters is being increasingly recognized . So far,IR occurs as part of a cluster of cardiovascular–metabolic abnormalities. Given the seriousness of the cardiovascular disease (CVD) data, there is an increasing burden of diabetes. In 2010, its global prevalence was estimated to be 6.4%. The incidence of diabetes among adults will rise between 2010 and 2030, as indicated by the 73% increase in the number of adults with diabetes in developing countries, compared to the 20% increase in developed countries . In thisTherefore, we asked the following research questions: (1) Are greater concentrations of thyroid hormones associated with IR in the total sample; (2) is a higher level of depression symptoms associated with IR in the total sample; and (3) do these associations differ between HOMA-IR? We tested these hypotheses using cross-sectional data from a randomized epidemiological study.The aim of the study was to investigate insulin resistance in association with thyroid function, psychoemotional state, and cardiovascular risk factors among 45–84-year-old citizens of Palanga.Data were available from a randomized epidemiological study performed in 2013. An independent random sample of 850 citizens of Palanga aged 45–84 years was drawn from the National Population Register. The potential respondents were contacted by phone. The research was accepted by the Lithuanian Bioethics Committee (protocol code: No. BE-2-25). Informed consent was obtained from all participants before the survey. Citizens of Palanga were selected as an object of investigation because they represent a close community with minor migration reflecting the population of the western part of Lithuania. There are no epidemiological data on IR, thyroid function, psychoemotional state, or cardiovascular risk factors in this region of Lithuania.All study participants were evaluated according to socio-demographic characteristics , behavioral factors, and self-perceived health using a questionnaire. Fasting blood samples were drawn from all participants, and biochemical tests were performed for the glucose, insulin, and thyroid hormone concentrations; total cholesterol; low-density lipoprotein (LDL); high-density lipoprotein (HDL); and triglyceride. The psychoemotional state was assessed using the Hospital Anxiety and Depression Scale (HADS)  and WHO Further inquiries regarding original questionnaires employed in the study can be directed to the corresponding author.The WHO-5 Well-being Index  questionThe HADS is a self-reported questionnaire with 14 items assessing symptoms of general anxiety (HADS-A) and depression (HADS-D) . Total sA questionnaire on general data  was usedArterial blood pressure (mmHg) was measured twice using a quicksilver sphygmomanometer on the right hand while a person was sitting, with the precision of 2 mm referring to the methodological recommendations . The ave2 (m) using the data from height and weight measurements. Overweight was diagnosed when BMI was 25.0–29.9 kg/m2, and obesity was diagnosed when BMI was 30.0 kg/m2 or more. Waist circumference was measured during the physician examination. Before the weight and height were measured, with participants standing without shoes or heavy outer garments, waist circumference was measured in the erect position at the midpoint between the lowest rib and the superior border of the iliac crest.Body height was measured in stockinged feet (without shoes) using a medical height rod. Body weight was measured without shoes using a medical scale. Body mass index (BMI) was calculated according to the formula BMI = body mass (kg)/heightThe standard questionnaire developed by Rose for the measurement of chest pain on exertion consists of eight test-type questions with possible answers . The Rosg and then frozen at –70 °C. The serum levels of the biochemical samples were analyzed via electrochemiluminescence immunoassay . Blood samples were analyzed for concentrations of glucose (mmol/L), norm (4.1–5.9); insulin (mU/L), norm (3.0–25.0); TSH (mIU/L), norm (0.55–4.78); FT4 (pmol/L), norm (11.5–22.7); FT3 (pmol/L), norm (3.5–6.5); thyroid peroxidase antibodies , norm (<60); total cholesterol (mmol/L), norm (2.6–5.2); LDL cholesterol (mmol/L), norm (2.6–3.4); HDL cholesterol (mmol/L), norm in men (0.9–1.7), norm in women (0.9–2.0); and triglyceride (mmol/L), norm (0.5–2.3). IR was calculated according to the HOMA-IR formula (HOMA-IR = (fasting plasma insulin [μIU/mL]) × (fasting plasma glucose [mmol/L])/22.5); the normal rate of HOMA is ≤2.7.Blood serum analyses were performed in a biochemical testing laboratory. The participants had been warned in advance to come having fasted for at least 12 h. Blood was taken from the elbow vein directly into vacuum blood collection systems, while the person was sitting (amount: 7 mL). The serum was separated from the blood by centrifugation at 3000× 2 test, the parametric two-tailed Student’s t-test, or nonparametric Mann–Whitney U test. The thyroid parameters  were divided into quartiles (Q1–Q4 (highest)) with the presence of MetS. The sensitivity, specificity, and predictive values were calculated for the HOMA-IR scores to define the best cut-off point when screening for possible T2DM. The area under the receiver performance characteristic (ROC) curve (AUC) was used as a summary measure of the capacity of the HOMA-IR to detect T2DM. Statistical analyses were performed using the Statistical Package for the Social Sciences software v.22 . The level of significance was set at p < 0.05.The clinical and sociodemographic characteristics are reported as frequencies and percentages for the categorical variables, with means and standard deviations for the continuous variables, and as medians (25th–75th percentiles) for variables with non-normal distribution. The similarity of the variable distribution to normal was assessed visually and using the Kolmogorov–Smirnov and Shapiro–Wilk tests. The data characteristics were compared between groups without HOMA-IR and with HOMA-IR using Fisher’s χn = 557) and with IR . Approximately 1.8% (n = 15) of the participants were excluded from the analysis due to a history of thyroid disease. In short, the mean age of the study population was 63.6 ± 10.3 years. The majority of participants  were female, and 67% (n = 557) were married. Of the participants, 68% (n = 565) had not obtained higher education.As demonstrated in The proportion of participants with bad well-being was 33%, as determined using the WHO well-being test; anxiety symptoms were shown by 33% of participants, and depression symptoms were shown by 20%, as measured by HADS. However, there were no significant differences in psychoemotional state between the study groups .As presented in No significant differences were found in the assessment of FT4, FT3, TSH, and anti-TPO between study groups in respect to IR .When the subjects were divided into groups according to symptoms of MetS, it was found that the TSH and FT3 concentrations and the FT3/FT4 ratio differed significantly between the groups. Significantly higher TSH and lower FT3 were found in the group with MetS. Not all patients in the group with MetS had IR (HOMA-IR > 2.7). Only 70.1% of subjects in the MetS group and 21.4% in the non-MetS group had IR .We correlated the thyroid parameters (divided into quartiles) with the presence of MetS. Subjects in the lowest (first) quartile for FT3 (<4.8 pmol/L) and FT3/FT4 ratio (<0.31) were significantly more likely to have MetS  compared with those in higher quartiles . There wThe HOMA-IR cut-offs for diagnosing T2DM are shown in The predictable accuracy is presented using ROC curves. The ROC curves for HOMA-IR scores in all subjects, and in women and men separately, are shown in The areas under the ROC curve using the HOMA-IR cut-off for diagnosing T2DM were 0.807 in the all-subjects group  0.779–0.834), 0.897 in men (95% CI 0.857–0.929), and 0.776 in women (95% CI 0.738–0.810).If individuals with IR have a high risk of diabetes or MetS, they are more likely to have depression even in normal and prediabetes conditions ,2,3,15. In terms of IR assessment, most of the foregoing studies assessed IR using HOMA-IR; however, various methods were used to assess depressive symptoms across the studies. In the present study, we used the HADS because of its well-established validity and reliability in evaluating depressive symptoms in various populations ,28. Our The relative importance of IR remains undefined as to the stratification of cardiovascular risk in the population, and the interactions between hyperglycemia, IR, and cardiovascular risk factors are complex and still underestimated . In our It is important to note that previous studies in the literature have detected several issues regarding the significant positive correlation between HOMA-IR and blood TSH levels ,41,42,43Furthermore, the findings indicate the presence of a HOMA-IR cut-off signaling established IR. This condition is one of the main pathogenetic mechanisms of T2DM among wealthy populations . The evaIn addition, it is important to note that the results of this study suggest the existence of HOMA-IR cut-offs that can predict the presence of T2DM, and HOMA-IR can be used not only to indicate the presence of IR. The introduction of this marker into clinical practice may help to prevent T2DM.In conclusion, an increase in fasting glucose and more frequent incidence of metabolic syndrome, diabetes, and cardiovascular diseases in subjects with IR are associated with the prevalence of cardiovascular risk factors.No significant association was found between thyroid function and HOMA-IR.Our data show the existence of HOMA-IR cut-offs that can predict the presence of T2DM, so HOMA-IR can be used not only to indicate the presence of IR. The introduction of this marker into clinical practice may help to prevent T2DM.


To investigate the expression of microRNA-122 (miR-122) in the progression of chronic hepatitis B virus- (HBV-) infected liver diseases, thus determining the role of serum miR-122 as a marker of HBV-caused liver injury. n = 63) and healthy volunteers (n = 11). And the serum miR-122 levels were detected using RT-qPCR. Moreover, an analysis was applied for identifying the specific correlation of the miR-122 level with HBV DNA, HBeAg, and ALT levels. After liver biopsy, Ishak scoring was utilized for evaluation of the fibrosis stage and the histological activity index (HAI).  Sera were collected from patients with different stages of HBV infection  and ALT . But no correlation of this molecule with HBeAg levels was found (P = 0.187). In comparison with the HBeAg-negative patients, serum miR-122 expression showed an increase in the HBeAg-positive patients (P = 0.001). miR-122 expression, in addition, was of a significant correlation with HAI, but not with the liver fibrosis score.  We confirmed, in the serum, increased miR-122 expression in HBV-infected patients and its highest expression in chronic HBV carriers, based on such comparison between the healthy controls and patients. The correlation analysis results were taken as confirmation of the positive relationship of miR-122 with HBV DNA ( The peak of the serum miR-122 expression normally occurs in the early stage of the progression from the HBV carrier phase to chronic hepatitis to cirrhosis. This molecule can be considered as a marker for evaluation of HBV-caused liver injury. Hepatitis B virus (HBV) infection is often mentioned as a serious public health issue which attacks 350-400 million people , 2. In cMicroRNAs (miRNAs) are noncoding RNAs with a length of about 21 to 23 nt and with high evolutionary conservation , 8. In 2Among the total hepatic miRNAs, approximately 70% are liver-specific miR-122 , 12. WanOn the basis of the above researches, we hypothesized that serum miR-122 can act to evaluate conditions HBV-induced liver injury at different stages. And there are no literature reports on the expression of miR-122 in different stages of HCC. Therefore, we analyzed the association of serum miR-122 with HBV DNA, ALT, and fibrosis degree. Furthermore, the comparison of levels of miR-122 at different stages of chronic HBV infection was carried out.n = 62) who underwent liver biopsy in the Infectious Diseases Ward of the Jingzhou Central Hospital from 2018 to 2019 and healthy volunteers (n = 11), the collected serum was anticoagulated with EDTA and stored at −80°C. Before serum collection, the informed consent signed by each subject was obtained. And an approval was gained from the Medical Ethics Committee of Jingzhou Central Hospital (JZCH2016006).On completion of the collection of serum from HBV-infected patients (Inclusion criteria: (1) control group: people who are confirmed healthy by recent physical examination. (2) HBV carrier group: people who are positive for HBsAg, HBeAg, and HBV DNA with persistently normal levels of serum ALT and AST and with no obvious abnormality shown in their liver biopsy. (3) CHB group: people who once suffered from hepatitis B or who had positive HBsAg for more than 6 months and positive HBsAg and/or HBV DNA till now and had a continual or repeated increase of ALT or who were found to have hepatitis lesions in their liver biopsy. (4) Cirrhosis group: people who were diagnosed as cirrhosis belonging to the Child-Pugh A grade by clinical images and/or liver biopsy.Exclusion criteria: (1) people with mixed infection of hepatitis B and C, alcoholic liver disease, fatty liver, HIV infection, drug-induced liver injury, autoimmune liver disease, or tumors. (2) People who once received or are receiving antiviral therapy.The COBAS AmpliPrep/COBAS TaqMan quantitative detection system , with 12 IU/mL as a detection limit, was utilized for determining the content of HBV DNA in serum following the reagent instructions. Meanwhile, the concentrations of HBeAg and HBsAg were measured by chemiluminescence microparticle immunoassay. Positive criteria for HBeAg: when the ratio of sample signal to cutoff (S/CO) < 1.000, the sample is considered as nonreactive; when the S/CO value ≥ 1.000, the sample is considered as reactive. The content of ALT was measured by the reduction coenzyme method.After the sectioning step of paraffin-embedded biopsied liver tissues, the staining step by hematoxylin-eosin (HE) and Masson was performed. The Ishak scoring system  was utilΔΔCt− method with U6 as an internal reference. The information of the probe primers synthesized by ABI Company is shown in Total RNA was separated from serum by utilizing the mirVana PARIS Kit . After measuring RNA concentration on a NanoDrop 2000 Nucleic Acid Analyzer , RNA was reversely transcribed into cDNA according to the instructions of the TaqMan MicroRNA Reverse Transcription Kit . Finally, RT-qPCR reactions were performed by the TaqMan MicroRNA Assay on a 7500HT real-time quantitative PCR instrument . The reaction was carried out with conditions including 95°C for 5 min and 40 cycles of 95°C for 15 s, 60°C for 60 s, and 72°C for 30 s. The relative expression of miR-122 was calculated using the 2H test was adopted, while Mann–Whitney U test for comparison between 2 groups. Correlation coefficients were detected using bivariable pairwise correlation analysis. Continuous variables were analyzed by the Pearson method, and categorical variables were analyzed by the Spearman method. P < 0.05 indicates that the difference is statistically significant.SPSS 17.0 software was used for statistical analysis. All experimental data were expressed as mean ± standard deviation (SD). For comparison among multiple groups, Kruskal–Wallis There were 73 serum samples, 62 from the HBV-infected patients and 11 from the control group. Baseline information about all subjects is described in P < 0.001). No marked differences between the CHB and cirrhosis groups in the miR-122 level was identified (P = 0.734)  .r = 0.354, P = 0.005). But no significant association between HBeAg and miR-122 was identified (P = 0.187) (n = 26), serum miR-122 expression showed an elevation in the HBeAg-positive patients (n = 36) (P = 0.001)   . Interes= 0.001) . Additio= 0.009) .r = −0.277, P = 0.030)  resulted in different miR-122 levels (P = 0.046)  (P = 0.699) was revealed  . Additio= 0.046) . In part= 0.199)  and degrrevealed .Association between HBV replication and miR-122 has been reported. When cells are infected with HBV, apoptotic or necrotic cells passively release miR-122 into the circulatory system. Some researchers believe that the release of miRNA into the blood may be one of the mechanisms by which cells adapt to the environment . In the It is generally believed that in comparison with HBV carriers, the degree of liver injury and miR-122 should be higher in CHB and cirrhosis patients. However, the opposite fact in this study is that in comparison with the CHB and cirrhosis groups, miR-122 expression markedly rose in the HBV carriers; there were no marked differences between the CHB and cirrhosis groups. That is to say, the maximum expression of serum miR-122 mainly occurs in the early stage of HBV infection-induced liver disease. Adding to that, domestic researchers have revealed that the changes of miRNA expression normally are seen in the early progression which HBV infection eventually develops into HCC . TherefoHBV DNA, as the most direct and reliable indicator of viral replication, is able to directly reflect the viral levels in vivo as well as the infectivity . In HBV-G < 3, 3 ≤ G < 6, G ≥ 6). Collectively, serum miR-122 can serve as a potential marker for evaluation of HBV infection-induced liver injury.ALT is a biochemical marker applied widely for evaluating conditions of liver injury. In the serum samples, we herein found a positive relationship between miR-122 and ALT, which is consistent with the foreign studies , 23. TheIn summary, we found that the peak of serum miR-122 levels normally occurs in the early stages of the progression from the HBV carrier stage to chronic hepatitis to cirrhosis. This provides a novel perspective and direction for mechanistic studies of HBV-induced disease. However, there were too many inadequate experimental samples in this study. More clinical samples are required to further investigate the expression of miR-122in HBV-infected liver disease.


P = 0.037) than survivors (n = 34), while the levels of the other biomarkers did not differ. Receiver operating characteristic analysis indicated that KL-6 shows the best performance for predicting survival . Multivariable Cox analysis that was adjusted by age and diffusing capacity for carbon monoxide suggested a high KL-6 level (> 53.5 pg/mL) as an independent prognostic factor for survival . Our results suggest that blood KL-6 might be a useful in predicting the prognosis for patients with SJS-ILD.Interstitial lung disease associated with primary Sjögren’s syndrome (SJS-ILD) has a variable clinical course. We aimed to investigate the role of blood biomarkers in predicting prognosis for SJS-ILD. Clinical data of 46 SJS-ILD patients were retrospectively reviewed. Plasma biomarker levels, including Krebs von den Lungen-6 (KL-6), CC chemokine ligand 18 (CCL18), chitinase-3-like-1 (YKL-40), interleukin-4 receptor alpha (IL-4Ra), and matrix metalloproteinase-7 (MMP-7) were measured using the multiplex Luminex assays . The median follow-up period was 69.0 months. The mean age of the patients was 59.4 years; 17.4% were men. The KL-6 level was significantly higher in non-survivors (n = 12; 119.6 vs. 59.5 pg/mL,  Lung involvement is a common extraglandular complication; the most frequent lung complication, interstitial lung disease (ILD), has a reported prevalence of 10–20%6. ILD has been reported to be associated with increased mortality in patients with SJS8. Although SJS-ILD generally follows a mild and self-limited course, it can exhibit a more severe and progressive course in some patients9. Therefore, it is important to find predicting factors that could differentiate those patients who will have the progressive disease from those that are expected to experience slow or stable disease, in order to provide appropriate intervention. Prognostic factors for patients with SJS-ILD, such as the baseline partial pressure of carbon dioxide and oxygen, forced vital capacity (FVC), the extent of reticular abnormality on high resolution computed tomography (HRCT), and the severity of fibroblastic foci, or the presence of microscopic honeycomb in a surgical lung biopsy have been reported previously as predicting factors for mortality in SJS-ILD12. However, the utility of these variables can be limited by insufficient effort on the part of the patient, reader variability in the interpretation of images, or invasiveness.Primary Sjögren syndrome (SJS) is a chronic systemic inflammatory disorder that is characterized by diminished function of the lacrimal and salivary glands13. The most-studied epithelial-specific biomarker in ILD is Krebs von den Lungen-6 (KL-6)15. KL-6 is a mucin-like glycoprotein that is of high molecular weight and is classified as a human MUC1 mucin protein. It is secreted by type II alveolar pneumocytes and bronchial epithelial cells in response to cellular damage16 and is therefore frequently considered an indicator for pulmonary damage and has been studied as a biomarker for disease activity in ILD18. The cytokines and enzymes that have been reported to be associated with the clinical course of IPF or CTD-ILDs include CC chemokine ligand 18 (CCL18), chitinase-3-like protein 1 (YKL 40), and interleukin-4 receptor alpha (IL-4Ra)22. CCL18 is a chemokine that is secreted mainly by alveolar macrophages (M2 phenotype)23 and is known to play an important role in the immune-mediated lung fibrosis processes in IPF24, and YKL 40 is a glycoprotein that is primarily secreted by macrophages, neutrophils, and certain types of local epithelial cells and is known to have a role in inflammation and tissue remolding25. A recent meta-analysis study revealed that serum YKL-40 is correlated with lung function and can therefore be used as a predictive biomarker for survival with IPF and CTD-ILD26. IL-4R mainly promotes the proliferation of T cells and B cells22, and can also stimulate the proliferation, differentiation, and activation of several other cell types including fibroblasts while also increasing the recruitment of inflammatory cells22. The extracellular matrix markers in ILD include matrix metalloproteinase-7 (MMP-7)29. The MMPs are a group of proteins that contribute to the activation of pro-fibrotic molecules, crucially contributing to tissue remodeling and fibrosis31. Of these, MMP-7 is reported to be associated with the severity and prognosis of IPF, suggesting its role in the fibrotic process28. However, the role of blood biomarkers in predicting prognosis in SJS-ILD is not well defined, and no study has yet compared the performance of various biomarkers in predicting SJS-ILD prognosis. Our study therefore aimed to compare the prognostic value of the different blood biomarkers to find the best biomarker for use in patients with SJS-ILD.On the contrary, blood biomarkers are relatively easy to test, independent of patient effort or reader ability. Biomarkers for idiopathic pulmonary fibrosis (IPF) or connective tissue disease (CTD)-ILD have mainly been investigated using proteins that are associated with epithelial damage, matrix turnover, or inflammation32. The study was approved by the Institutional Review Board of Asan Medical Center (2018-1115), and written informed consent for the use of the blood samples for clinical research was obtained from all patients. All methods were performed in accordance with the relevant guidelines and regulations.A total of 62 patients diagnosed with primary SJS-ILD  between January 2000 and December 2016 at Asan Medical Center, Seoul, Republic of Korea, were screened in this study. Of these, only subjects with available blood samples were finally included in this study (n = 46). The patients excluded from the cohort showed lower C-reactive protein level than those included  and the diffusing capacity of the lung for carbon monoxide (DLCO) were measured according to the recommendations from the American Thoracic Society (ATS)/European Respiratory Society (ERS). The results were presented as percentages of the normal predicted values36. A six-minute walk test (6MWT) was performed according to ERS/ATS recommendations37, and bronchoalveolar lavage (BAL) was performed according to the ATS guildelines38. Data from follow-up assessments at 3–6-month intervals or from hospitalization events were reviewed to determine the development of acute exacerbation (AE). AE was defined according to the criteria suggested by Collard et al.39, which is worsening dyspnea within 30 days with new bilateral lung infiltration with no evidence of infection or other alternative causes for the dyspnea .The clinical and survival data of all patients were retrospectively collected from medical records, and/or the records of the National Health Insurance Service of Korea. To measure disease activity in patients with primary SJS, EULAR Sjögren’s Syndrome Disease Activity Index (ESSDAI) scores at the time of ILD diagnosis was calculatedBlood samples were obtained at the time of diagnosis by venipuncture and immediately centrifuged. The separated plasma samples were then stored at − 80 °C until biomarker measurement. The plasma levels of KL-6, CCL18, YKL-40, IL-4Ra, and MMP-7 were measured using the multiplex Luminex assays  in accordance with the manufacturer’s instructions.3. A UIP pattern was defined by a subpleural and basal predominance of reticular abnormalities, honeycombing with or without traction bronchiectasis, and the absence of inconsistent findings with a UIP pattern such as extensive ground-glass opacities (GGO), micro-nodules, discrete cysts, or segmental/lobar consolidations40.HRCT scans were obtained in accordance with standard protocols and reviewed by a radiologist (J.C.) who was blinded to the clinical information. The HRCT patterns were categorized into usual interstitial pneumonia (UIP), probable UIP, indeterminate for UIP, or an alternative diagnosis based on the 2018 Fleischner Society guidelinest-test or Mann–Whitney U test were used for the continuous data, while the Pearson’s chi-squared or Fisher’s exact test were utilized to analyze the categorical data. ROC curve analysis was performed to evaluate the optimal cut-off value of blood biomarkers for predicting survival. The risk factors for mortality were evaluated using a Cox proportional hazard model. Due to the limited number of death events, among variables with P < 0.1, age, and DLCO, which were previously prognostic factors in SJS-ILD42. were used as adjustment variables for the multivariable Cox analysis. Survival was evaluated using Kaplan–Meier survival analysis and the log-rank test. Survival time was calculated from the date of ILD diagnosis to death or censoring, which took place on December 31, 2016 and included all patients who were still alive on this date. Spearman’s rank correlation coefficients were performed to evaluate the correlation between blood KL-6 levels and lung function or exercise capacity. All P-values were two-tailed with statistical significance set at a P value of < 0.05, and all statistical analyses were performed using SPSS Statistics, Version 24.0. .All values were expressed as mean ± standard deviation for continuous variables or percentages for categorical variables. The Student’s The mean age of the 46 patients with primary SJS-ILD was 59.4 years and 17.4% were males Table .Table 1CThe non-survivors had lower TLC and lymphocyte levels in their BAL fluid, and showed positive anti SS-A/Ro and a UIP pattern on the HRCT more frequently than the survivors Table . Total oP = 0.037) as compared to the survivors (Table P = 0.037) using ROC analysis for 10-year survival, and the optimal cut-off value was 53.5 pg/mL  .The level of KL-6 was significantly elevated in the non-survivors , a UIP pattern on the HRCT, and a higher level of KL-6 (> 53.5 pg/mL) were significantly associated with 10-year mortality  was independently associated with a poor prognosis   Table . HoweverCO, TLC) and poorer exercise capacities  in the high KL-6 group  than the low KL-6 group  (Table P = 0.017) than the low KL-6 group  and were less likely to survive  than those with low KL-6 levels. , DLCO , and 6MWD   Fig. . HoweverCO. Patients with high KL-6 levels showed poorer lung function, with more frequent AE and death than those with low KL-6 levels. Moreover, KL-6 levels were found to correlate with the severity of the disease in patients with SJS-ILD.In this study, the prediction of survival using various biomarkers was evaluated for patients with primary SJS-ILD. We focused on evaluating whether biomarkers that were previously studied in other CTD-ILDs are useful in patients with SJS-ILD in terms of the severity and prognosis for ILD. KL-6 was found to be superior to CCL18, YKL-40, IL-4Ra, and MMP-7 in predicting the prognosis of patients with SJS-ILD. High KL-6 levels were independently associated with an increased risk of 10-year mortality in patients with SJS-ILD when adjusted by age and DLCO when using multivariable Cox analysis. These findings are consistent with those in previous reports19. Kamiya et al. studied 99 patients with SJS-ILD (with a median follow-up period of 5.97 years) and reported that a higher level of KL-6 (> 800 U/mL) was associated with poor survival  using multivariable Cox analysis adjusted by age and gender12. Kim et al. also showed that a high KL-6 level (≥ 640 U/mL) was an independent prognostic factor for survival  in 158 patients with rheumatoid arthritis (RA)-ILD. These findings suggest that KL-6 might be useful for predicting the clinical outcomes of CTD-ILD, including SJS-ILD. In addition to identifying the usefulness of KL-6 for predicting the prognosis of patients with SJS-ILD, our study revealed that KL-6 showed better performance for predicting survival compared to other biomarkers.KL-6 showed the best performance in predicting 10-year mortality as compared to the other biomarkers. A high KL-6 level (> 53.5 pg/mL) was found to be an independent risk factor for mortality after adjustment for age and DL43. Ohshimo et al. studied 77 patients with IPF and reported that a high KL-6 level (≥ 1300 U/mL) was an independent risk factor for the development of AE  when adjusted for age, sex, smoking history, and treatment43. They also showed that baseline KL-6 levels were significantly higher in patients who experienced AE than those who did not 43. Lee et al. studied 165 patients with CTD-ILD , and also showed that the semiquantitative grades of ILD on the HRCT  were significantly proportional to serum KL-6 levels, from which grades could be successfully differentiated 17. They also showed that serum KL-6 level had a moderate negative correlation with both FVC%  and DLCO% 17.In this study, patients with high KL-6 levels experienced AE more frequently, and KL-6 levels were negatively correlated with lung function and exercise capacity, suggesting the value of KL-6 for predicting AE and disease severity. Previous reports support our findings29, with several reporting these biomarkers useful in the prognosis of CTD-ILD. Tiev et al. studied 83 patients with systemic sclerosis-ILD, and reported that a high baseline CCL18 level (> 187 mg/mL) was a predictive factor  for worsening of the subsequent disease (with a decrease of > 10% predicted in FVC or TLC) within 2 years using multivariable Cox analysis20. Hozumi et al. also demonstrated a correlation between serum YKL-40 levels and a lower arterial oxygen pressure  in 72 patients with polymyositis/dermatomyositis-ILD, and independent association with poor prognosis under multivariable Cox analysis when adjusted by the anti-aminoacyl tRNA synthetase status  or the anti-CADM-140/melanoma differentiation-associated gene 5 antibody status 21. Moreover, Nakatsuka et al. studied 52 patients with polymyositis/dermatomyositis-ILD, and showed that higher levels of serum MMP-7 were associated with 6-month mortality  using univariate logistic regression analysis, and that high serum MMP-7 (> 5.08 ng/mL) was associated with a worse prognosis  when using multivariable logistic regression analysis adjusted by hypoxia and serum ferritin levels29. Due to these inconsistent results, the prognostic value of the above markers is not clear for SJS-ILD. Further verification on a larger scale is required for clinical use in patients with SJS-ILD.In our study, CCL18, YKL-40, IL-4Ra, and MMP-7 were not found to be associated with the prognosis or severity of ILD. However, previous studies have reported contradictory findingsP = 0.329), anti-SSA , and anti-SSB  was not different between the non-survivors and survivors, suggesting that autoantibodies were not associated with prognosis of ILD44. However, there is also a contradictory report; Boitiaux et al., in 45 newly diagnosed patients with idiopathic interstitial pneumonia, showed that the anti-SSA (+) group (n = 15) had lower vital capacity  and more frequent GGO  and reticulation  on HRCT than the anti-SSA(−) group (n = 30)45. Due to these inconsistent findings, it is still insufficient to draw a conclusion about the association between autoantibodies and prognosis of SJS-ILD.In this study, positive anti-SSA was more frequent in survivors but was not associated with prognosis in the unadjusted Cox analysis. Moreover, there was no difference in the frequency of positive autoantibodies such as ANA and anti-SSB between the non-survivors and survivors. The previous study support our findings; Gao et al., in 178 patients with SJS-ILD, reported that the frequency of positive ANA (65.2 vs 75.8%, 17. Second, we did not include a validation cohort to confirm our findings. Our sample size was relatively small; however, the rarity of SJS-ILD means that the number of patients studied is not considered low, and the long-term observation in our study can provide important insights into this rare condition. Finally, detailed treatment information such as type, dose, timing, and the duration over which medication was given were not considered in the analysis of the prognostic factors. However, the treatment given to survivors and non-survivors did not differ.Our study has some limitations. First, this was a retrospective study conducted at a single center, which might lead to selection biases or a lack in generalizability. However, the baseline characteristics of our patients were comparable to those in other studiesIn conclusion, our results suggest that blood KL-6 might be a useful predictor of the prognosis for patients with SJS-ILD.Supplementary Information.


This study aimed to investigate the effects of d-allulose on aerobic performance and recovery from exhaustion and compared them with the effects of exercise training. Male C57BL/6J mice were subjected to exercise and allowed to run freely on a wheel. Aerobic performance was evaluated using a treadmill. Glucose metabolism was analyzed by an intraperitoneal glucose tolerance test (ipGTT). Skeletal muscle intracellular signaling was analyzed by Western blotting. Four weeks of daily oral administration of 3% d-allulose increased running distance and shortened recovery time as assessed by an endurance test. d-Allulose administration also increased the maximal aerobic speed (MAS), which was observed following treatment for >3 or 7 days. The improved performance was associated with lower blood lactate levels and increased liver glycogen levels. Although d-allulose did not change the overall glucose levels as determined by ipGTT, it decreased plasma insulin levels, indicating enhanced insulin sensitivity. Finally, d-allulose enhanced the phosphorylation of AMP-activated protein kinase and acetyl-CoA carboxylase and the expression of peroxisome proliferator-activated receptor γ coactivator 1α. Our results indicate that d-allulose administration enhances endurance ability, reduces fatigue, and improves insulin sensitivity similarly to exercise training. d-Allulose administration may be a potential treatment option to alleviate obesity and enhance aerobic exercise performance. The global epidemic of obesity and obesity-associated diseases, especially type 2 diabetes, is expected to continue to worsen . Exercisd-Allulose, also known as d-psicose, is a rare, functional sugar formed by the epimerization of d-fructose at the C-3 position [d-Allulose has been shown to ameliorate insulin resistance [position . d-Allulsistance ,6,7 and sistance ,9,10 andsistance  and redusistance ,13,14 ansistance ,16.d-allulose would enhance exercise performance because its administration has been shown to increase muscle or liver glycogen storage and fat oxidation. Matsuo reported that daily intake of d-allulose may accelerate the repletion of liver and muscle glycogen after exhaustive swimming [d-allulose enhanced the translocation of liver glucokinase and increased liver glycogen content [d-allulose promoted fat oxidation [d-allulose intake has also been reported in healthy humans [d-allulose on aerobic performance have not been reported. Therefore, in the present study, we assessed whether d-allulose administration improves endurance ability and accelerates recovery from exhaustion. We further compared the effects of d-allulose with exercise training effects to understand its mechanism of action.ATP availability is essential for skeletal muscle contraction in sprint events (anaerobic) as well as endurance events (aerobic) . Both faswimming . Long-te content . In addixidation , and enhy humans . To the d-allulose diet . The composition of the experimental diets is presented in d-Allulose was provided by Matsutani Chemical Industry Co. Ltd. . To standardize the number of calories in the diets, d-allulose was replaced with cellulose. A free-wheel running apparatus  was placed into individual cages housing mice assigned to exercise, and the mice were allowed to run freely on the wheels. Food intake was measured once every 2 days. Body weights were measured weekly. All experimental procedures involving animals followed the guidelines for the Care and Use of Laboratory Animals of Nagoya University. Ethical approval was granted by the Animal Experiment Committee of Nagoya University (HPFS No. 20).Six-week-old male C57BL/6J mice were purchased from SLC Co. Ltd. . The mice were singly housed in cages in a room maintained at 23 ± 2 °C under a 12 h light/12 h dark cycle and allowed free access to a standard chow diet  and water from the period of environmental adaptation at week 1 until the beginning of the experiment. During the intervention period, mice were either kept on the chow diet  or a n = 6) and a d-allulose diet group . Running wheels were placed in all cages. As shown in d-allulose diet. Following treadmill acclimatization (described later), 6-week-old mice underwent adaptation for 1 week and treadmill acclimatization for 3 weeks. Mice were randomly assigned into two groups: a chow diet group , sedentary/d-allulose group , exercise/chow diet group , and exercise/d-allulose group . To standardize initial endurance ability, the mice were grouped based on the lactate levels immediately after running on a treadmill for 2 h at 20 m/min (the first BGL test). Mice in the exercise groups (E2 and AE2) had free access to the wheel after the grouping at the start of week 3. As shown in Ten-week-old mice were divided into four groups: sedentary/chow diet group  were on an alternate feeding regimen of chow diet or d-allulose. We measured both glucose and lactate with a biosensor BF-5S instrument  before and after each maximal aerobic speed (MAS) test, performed as shown in Ten-week-old mice . Mice were first accustomed to the treadmill by running at 5 m/min for 10 min (warm-up), then speeding up to 10 m/min for 30 min, three times, and then the speed was increased to 20 m/min. Mice were encouraged to run by light electrical stimulation (0.2 mA) provided by a grid located at the rear end of the treadmill belt. The wireless running wheel apparatus  was used to monitor the movement of the mice. Rotation of the wheel by mice transmitted a wireless electronic signal to a hub, and the number of revolutions was recorded on Wheel Manager software (Med Associates Inc.) every few seconds. Activity was recorded as rotation over time, and the data were exported to a Microsoft Excel spreadsheet . We followed the protocol by Pauly et al. , and conAerobic endurance was determined as the distance that a mouse could move when it reached a state of exhaustion at a moderate-intensity running speed of about 75% MAS . After aThe recovery of mice from a maximum exercise load was evaluated by comparing the change in wheel running distance after the endurance tests. The recovery ratio was defined as the ratio of daily exercise volume for each mouse to the average daily running distance during the 7 days before the endurance test. The percentage of baseline activity  was usedBlood was obtained from the tail veins of mice. After a 10-minute warm-up at 5 m/min, the speed was increased by 2 m/min every 2 min until 20 m/min and then maintained for 2 h. Tail blood was collected and used to determine blood glucose and lactate levels before running  and immediately after running (0 min).g for 10 min at 4 °C) of blood, stored at −80 °C, and used to determine insulin levels with the mouse ELISA KIT  according to the manufacturer’s instructions.Mice were subjected to fasting for 4 h before the test. The running wheels of the E2 and AE2 groups were removed 4 h before the ipGTT to avoid an acute effect of voluntary exercise. Each mouse received an intraperitoneal injection of glucose solution (2 g/kg BW), and blood samples were collected at 0, 15, 30, 60, and 120 min after the injection. Blood glucose levels were determined using the biosensor BF-5S instrument. Blood was also collected in Eppendorf tubes containing heparin (1:1000) at 15 min. Plasma was obtained following centrifugation  according to the manufacturer’s instructions.g for 30 min, 4 °C). We performed Western blotting using a previously described protocol [Proteins in the soleus muscle were extracted using a homogenization buffer . The supernatants containing protein samples were obtained after centrifugation (7000× t-test and one-way ANOVA (using Tukey’s or Dunnett’s post hoc test) on Prism 8 software . Differences at p < 0.05 were considered as being statistically significant.Results are shown as mean ± standard error of the mean (SEM). Data were analyzed using Student’s d-allulose group than in the control group ; E2 group 23.6% (p < 0.01); AE2 group 24.8% (p < 0.01); The MAS of mice in the C2 group decreased by approximately 10%, while that of mice in the other groups increased significantly (A2 group 12.7% (p < 0.001). Blood glucose and lactate levels before and immediately after running for 2 h at 20 m/min are shown in d-allulose or exercise.IpGTT profiles were similar among the four groups, except at 15 min A, and noAs shown in p = 0.06). The muscle glycogen level increased in the E2 and AE2 groups but not in the A2 group -induced obese rodents, db/db mice, type 2 diabetes model rats, and humans with borderline diabetes [d-allulose on aerobic exercise performance have not been previously reported. Interestingly, d-allulose administration increased running distance and running speed and induced a rapid recovery from exhaustive running. Furthermore, d-allulose administration improved insulin sensitivity and induced changes in signaling molecules that were similar to those observed with exercise.In the present study, we investigated the effect of diabetes ,9,10,11.d-allulose on aerobic exercise performance can depend on the availability of carbohydrates and fat, which are the main sources for ATP production. Glycogen contents in the muscles and liver are associated with endurance and recovery from exhaustive exercise. Although muscle glycogen is essential for ATP production during exercise in humans [d-allulose. These effects of d-allulose may be due to increased levels of liver glycogen. However, d-allulose can also impact exercise performance through changes in fat oxidation.The effects of n humans , the conn humans . The pron humans . Pederson humans . Recentln humans . In our d-allulose administration enhanced the AMPK axis, which is consistent with d-allulose enhancing the oxidation of fatty acids [d-allulose administration has been demonstrated in rats [Lipids are another primary source for the production of ATP. During muscle contraction, the uptake of fatty acids in skeletal muscle increases, while the activity of ACC decreases, which promotes the phosphorylation of ACC . PGC-1α ty acids ,25. A stty acids . An incr in rats  and humad-allulose administration or exercise training. As the intensity of endurance running was likely to be below the lactate threshold levels [d-allulose effectively reduced lactate levels after running. As lactate levels are expected to increase if increased liver glycogen is driving the improvement in MAS, we speculate that the improvement in MAS can be, at least in part, because of improved fat oxidation. Blood glucose levels significantly decreased during the period of chow diet administration but not during the d-allulose administration period, which suggests that d-allulose administration increased glycogen levels or the preferred use of free fatty acid. The accumulation of blood lactate indicates the aerobic/anaerobic transition and is an important marker for endurance exercise capacity . Lactated levels , the difd-allulose administration. Metabolic adaptation  has been reported to occur even before mitochondrial changes [d-allulose may exert acute and chronic effects on exercise performance. The acute effect may be associated with its effect on glycogen levels. A previous report [d-allulose administration can alter glycogen levels in muscle or the liver and aerobic performance. In contrast, muscle adaptation induced by d-allulose administration, accompanied by changes in the expression of AMPK, ACC, and PGC-1α, takes time. Studies have focused on the role of AMPK as a mediator of cell signaling pathways related to skeletal muscle function and metabolism [Mitochondrial changes accompanied improvements in fat oxidation. Activation of AMPK-PGC-1α signaling induces mitochondrial synthesis. It remains unclear whether these adaptations in muscle metabolism occur within a few days of  changes ,41. Simis report  and our tabolism .d-Allulose reduces visceral fat and body weight and improves insulin sensitivity. Iwasaki et al. [d-allulose administration reduced food intake mediated by GLP-1 release and vagal afferent activation. d-Allulose administration also increases energy consumption [d-allulose administration reversed insulin resistance induced by a high-sucrose [d-allulose administration in both experiments, which reduced food intake in the high-sucrose diet group but not in the high-fat diet group. In the present investigation, d-allulose administration reduced both food intake and body weight. i et al.  reportedsumption . We prev-sucrose  and high-sucrose  diet. Ind-allulose administration did not affect food intake and body weight in the exercise groups. Further research is needed to determine how exercise is associated with the effects of d-allulose on food intake or energy expenditure. The ipGTT results indicated that d-allulose administration or exercise enhances insulin sensitivity, suggesting that exercise and d-allulose affect insulin sensitivity through different mechanisms.Interestingly, d-allulose has been proposed as a caloric restriction mimetic [d-allulose as an exercise mimetic, which shares common features with caloric restriction mimetics. The present data indicated that d-allulose can prevent fatigue during or after exercise. The limitation of our study is the lack of direct evidence that improved FFA utilization contributes to the changes in aerobic performance. We also used only male mice, and sex-specific differences in the effect of d-allulose were not tested. To draw conclusions on the effect of d-allulose on insulin sensitivity in normal mice, an additional study using a hyperinsulinemic–euglycemic clamp method is necessary. Further research with human subjects is warranted to explore the anti-fatigue effects of d-allulose. Nevertheless, our study not only provides valuable insights into the potential role of d-allulose in alleviating obesity and enhancing aerobic exercise performance in humans but also conclusively establishes a viable and promising option to ameliorate the clinical outcomes of obesity-induced health problems by using d-allulose.Owing to its chronic effect on body weight and insulin resistance,  mimetic ,45,46. W